Abstract
We report about a new intrinsically calibrated sensor technology exemplified by means of an O 2-FCSensor. The O 2-FCSensor is particularly suited for the quantitative determination of molecular oxygen within biological gases and fluids. The technology is based on the determination of the highly O 2-dependent triplet transition rates of suitable fluorophores such as Rhodamine Green or Fluorescein. The method utilizes fluorescence correlation spectroscopy (FCS) with the ultimate detection sensitivity of a single fluorescent molecule. Calibration curves of the triplet fraction ( T) and triplet relaxation time ( τ T ) as a dependence of the oxygen concentration have been determined using a thin (μm) sensor layer, and can be well approximated by hyperbolic sensor characteristics with oxygen sensitivities in the order of 0.1/(%O 2). Interestingly, the oxygen sensitivity of triplet fractions of O 2-FCSensors are not influenced by changes in viscosity of the solution demonstrating that FCS-based O 2 measurements are not diffusion controlled (in contrast to the dynamic process of collisional fluorescence quenching by molecular oxygen). Due to the ultimate sensitivity of this technique it can be applied to nanomolar or even single molecule concentrations of fluorophores thus toxic or cancerogenic influences of the applied indicators are avoided or at least minimized. Furthermore, new fluorescent indicator classes with exceptional photophysical properties can be explored for FCS-based chemo- and biosensors enabling the measurement of other analytes such as, e.g. hydrogen ion activities and carbon dioxide. As a consequence, blood gas analysis (pH, pCO 2 and pO 2) may be realized on the basis of intrinsically calibrated FCS-Sensor technology comprising the potential of absolute measurement with unprecedented sensitivity.
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